Exploring chromatin organization and transcription in S. pombe and hematopoietic development

Abstract

DNA in the eukaryotic nucleus is organized into histone-DNA complex, so-called chromatin, through forming an array of nucleosomes. Each nucleosome consists of a 147bp DNA wrapped around a histone octamer harboring two of each H2A-H2B and H3-H4. Chromatin is orderly packed several times forming a chromosome structure. Active euchromatin and repressive heterochromatin are defined according to the degree of DNA compaction, of which euchromatin is open, and heterochromatin is condensed. Chromatin organization and its regulation always affect downstream gene transcriptions through different mechanisms, which consequently play crucial roles in many cellular and biological processes.

In this thesis, we explored mechanisms of chromatin organization and its associated regulatory factors by using Schizosaccharomyces pombe. We identified an uncovered role of Abo1 in different heterochromatin locus. We demonstrated that Abo1 is involved in Clr4 mediated heterochromatin assembly through regulating H3K9me2 to H3K9me3 transition, related to distinct silencing machinery.

We also performed multiple in vitro experiments to investigate the functional role of the chromatin remodeler Hrp3, which is the orthologue of human CHD1. We generated several mutant strains where the non-catalytic domains were individually deleted. Our result suggested non-catalytic domains could further affect ATP hydrolyzing activity, and may further affect the chromatin remodeling function.

In this thesis, we also investigated the outcomes of epigenetic and transcriptional regulation in hematopoietic development. We performed analysis on CAGE libraries in various primary cell types from the Fantom 5 project to study the usage of alternative transcriptional start site (TSS). Through mapping the TSS to Refseq, we identified alternative TSS that can lead protein domain loss. The alternative TSSs were shown to be expressed at different levels in different cell types or developmental stages, particularly in blood cells. We further investigated the functional consequence of alternative TSSs usage for KDM2B in Jurkat T-cells.

To identify critical novel epigenetic regulators for myeloid differentiation, we performed a CRISPR-cas9 screen. We identified the chromatin remodeler CHD2 as a crucial regulator for megakaryocyte differentiation in the PMA inducible K-562 cell model.